WO2003076927A1 - Procede et appareil pour determiner un descripteur moleculaire d'absorption pour un candidat compose - Google Patents

Procede et appareil pour determiner un descripteur moleculaire d'absorption pour un candidat compose Download PDF

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Publication number
WO2003076927A1
WO2003076927A1 PCT/US2003/006756 US0306756W WO03076927A1 WO 2003076927 A1 WO2003076927 A1 WO 2003076927A1 US 0306756 W US0306756 W US 0306756W WO 03076927 A1 WO03076927 A1 WO 03076927A1
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Prior art keywords
absorption
membrane
poly
molecular
descriptors
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PCT/US2003/006756
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English (en)
Inventor
Jim E. Riviere
Xin-Rui Xia
Ronald E. Baynes
Nancy A. Monteiro-Rivier
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North Carolina State University
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Priority to AU2003228280A priority Critical patent/AU2003228280A1/en
Publication of WO2003076927A1 publication Critical patent/WO2003076927A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
    • G01N2013/003—Diffusion; diffusivity between liquids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N2030/009—Extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Definitions

  • the present invention relates to a method and apparatus for assessment of membrane absorption, and in one embodiment to a method and apparatus that employs a fiber coated with a skin-imitating membrane and an absorption container for rapid assessment of percutaneous absorption.
  • a key function of skin is to provide a barrier that protects the body from foreign substances. Any drug or chemical agent must penetrate the skin's barrier to act ether locally or systemically. Decades of study have established that skin comprises two layers, epidermis and dermis. The epidermis has no capillary blood flow but is made up of several layers of enzymatically active cells, while the dermis in the skin inner layer contains the capillary network that transports the drug or chemical agent to the systemic circulation.
  • Skin-imitating membranes differ advantageously from human skin epidermis due to their ready availability, uniformity, tensile strength and chemical purity. Feldstein, M.M.. et al.. J. Controlled. Release 52 (1998) 25-40; Bavnes, R. E., et al.. Toxicology Industrial Health 16(2000) 225-233. Silastic membranes are the most widely used skin-imitating membranes because of their high permeability comparable with human stratum corneum; and its properties can be modified to simulate skin. There are mainly two kinds of diffusion chambers currently found in the art, Franz diffusion cell (A) and flow-through diffusion cell (B) as shown in Figure 1.
  • Franz diffusion cell A
  • B flow-through diffusion cell
  • the membrane (a) is placed between two chambers, donor (b) and receptor (c), and the compound in question diffuses from the donor phase through the membrane into the receptor phase.
  • Franz diffusion cell samples are withdrawn periodically from the receptor phase (g) and analyzed to measure the penetration flux.
  • the compounds passing through the membrane are carried away by the receptor fluid flowing beneath the membrane undersurface to be collected in discrete volumes at a remote location.
  • the advantages of the flow-through cell are allowing automatic sampling; maintaining sink conditions since the receptor fluid is replaced continuously, and mimicking the subcutaneous blood flow by the movement of the receptor fluid beneath the undersurface of the membrane. Bronaugh, R.L. (ed.), Percutaneous absorption: drugs-cosmetics-mechanisms- methodology.
  • the concentration of a given compound is C° in the bulk solution of the donor phase. In the vicinity of the membrane the concentration of the compound is lower than the bulk solution because of the absorption by the membrane, which results in a concentration gradient (C° ⁇ C dX ) in the boundary layer between the membrane and the donor phase. There is also a concentration gradient (Crx ⁇ Cr) in the boundary layer between the membrane and the receptor phase because the compound passing through the membrane is carried away by the receptor fluid.
  • the surface concentration in the receptor phase (Crx) is also a relevant concentration for the penetration. It is this available concentration that determines the diffusion rate into the receptor phase rather than the concentration in the membrane.
  • a method of determining a molecular descriptor of absorption for a candidate compound comprises:
  • a method of determining a molecular descriptor of absorption for a candidate compound comprises: (a) providing a test system comprising: (i) a membrane assembly comprising one of a fiber and a simulated biological membrane membrane disposed thereon and a tube and a simulated biological membrane disposed therein; and (ii) a container comprising a cover having one or more apertures disposed therein, the one or more apertures adapted to receive the membrane assembly; (b) providing a test solution in the container, the test solution comprising one or more candidate compounds, wherein the one or more candidate compounds are present in a known concentration; (c) contacting the test solution with the simulated biological membrane by placing the membrane assembly into an aperture of the container cover, ,whereby the one or more candidate compounds partition into the membrane; (d) detecting the presence or amount of the one or more candidate compounds in the membrane at one or more permeation times; and (e) determining a molecular descriptor of absorption using the presence or amount of the one or more
  • the method comprises determining a plurality of molecular descriptors of absorption.
  • the test solution can comprise a plurality of test compounds, e.g. a plurality of different test compounds.
  • the method can comprise contacting the test solution with two or more simulated biological membranes to partition the one or more candidate compounds into the membranes.
  • the two or more simulated biological membranes can be the same or different.
  • the test solution can be stirred during the contacting of step (b).
  • the membrane can be configured by one of disposing the membrane on a fiber or disposing the membrane within a tube.
  • the membrane has a constant thickness along the fiber.
  • the simulated biological membrane can simulates a biological barrier or membrane selected from the group consisting of subcellular, cellular, oral/mucosal, gastrointestinal, blood-brain, respiratory- lung, nasal, ocular, subconjuctival, and skin.
  • the detecting is done by gas chromatography or high performance liquid chromatography, such as by injecting the fiber into a gas chromatograph or high performance liquid chromatograph.
  • a molecular descriptor of absorption can be compared to a reference molecular descriptor of absorption.
  • a system for determining a molecular descriptor of absorption for a candidate compound comprises: (a) a membrane assembly comprising one of a fiber and a simulated biological membrane membrane disposed thereon and a tube and a simulated biological membrane disposed therein; and (b) a container comprising a cover having one or more apertures disposed therein, the one or more apertures adapted to receive the membrane assembly.
  • the system can comprise a stirring platform adapted to receive the container and a stir bar for use therewith.
  • the system can comprise an apparatus for quantitatively analyzing the presence or amount of one or more candidate compounds in the membrane.
  • the apparatus for quantitative analysis can be a gas chromatograph or a high performance liquid chromatography apparatus.
  • a method of assessing susceptibility of a candidate compound to absorption into a biological membrane is also disclosed.
  • the method comprises: (a) obtaining a molecular descriptor of absorption for a candidate compound in one or more simulated biological membranes, wherein the one or more simulated biological membranes simulate the biological membrane; (b) comparing the molecular descriptor of absorption to a reference molecular descriptor of absorption; and (c) assessing susceptibility to absorption into the biological membrane based on the comparing of step (b).
  • the molecular descriptor of absorption can be compared to a plurality of reference molecular descriptors of absorption.
  • a plurality of molecular descriptors of absorption for the candidate compound can be obtained.
  • the plurality of molecular descriptors of absorption can be compared to a plurality of reference molecular descriptors of absorption.
  • the one or more simulated biological membranes can simulate a biological barrier or membrane selected from the group consisting of subcellular, cellular, oral/mucosal, gastrointestinal, blood-brain, respiratory-lung, nasal, ocular, subconjuctival, and skin.
  • a computer-readable medium having stored thereon instructions for assessing susceptibility of a candidate compound to absorption into a biological membrane is also disclosed.
  • a system for assessing susceptibility of a candidate compound to absorption into a biological membrane comprises: (a) an input for obtaining a molecular descriptor of absorption for a candidate compound in one or more simulated biological membranes, wherein the one or more simulated biological membranes simulate the biological membrane; (b) a database for comparing the molecular descriptor of absorption to a reference molecular descriptor of absorption; and
  • the system can comprise a database for comparing the molecular descriptor of absorption to a plurality of reference molecular descriptors of absorption.
  • the system can comprise an input for obtaining a plurality of molecular descriptors of absorption for the candidate compound.
  • the system can comprise a database for comparing the plurality of molecular descriptors of absorption to a plurality of reference molecular descriptors of absorption.
  • a computer-readable medium having stored thereon a data structure, comprising: (a) a first data field containing data representing a type of a molecular descriptor of absorption; and (b) a second data field containing data representing a value of a molecular descriptor of absorption.
  • FIGS 1A andl B are diagrammatic representations of the Franz diffusion cell and flow-through cell, respectively.
  • Cell components are as follows: (a) membrane; (b) donor compartment; (c) receptor compartment; (d) water jacket; (e) receptor inlet; (f) receptor outlet; (g) receptor sampling port;
  • C dX surface concentration in the donor phase
  • C mr surface concentration in the membrane contacting the receptor phase
  • C rx surface concentration in the receptor phase
  • C r bulk concentration in the receptor phase
  • ⁇ d thickness of the boundary layer of the donor phase
  • ⁇ r thickness of the boundary layer of the receptor phase
  • ⁇ m Thickness of the membrane.
  • D d , D m , and D r are the diffusion coefficients in the donor phase, in the membrane, and in the receptor phase, respectively.
  • Figure 3 depicts the membrane-coated fiber assembly of the present invention.
  • Figure 4 depicts a system of the present invention, including container with needle holding cap and water jacket.
  • Figure 5 depicts a system of the present invention, including absorption setup with coated fibers.
  • Figure 6 shows GC/MS spectra acquired with a HP 5890 gas chromatograph coupled with a HP 5970B mass selective detector and a polydimethylsiloxane (PDMS) membrane coated fiber partitioned for 30 minutes in a solution containing 30 compounds.
  • the 30 compounds were: 1 : Terrazole,
  • Figure 7 shows the absorption amount versus time profiles for three compounds, Dacthal, Heptachlor Epoxide and tr-Nonachlor, and their regressions.
  • Figure 12 is a plot of physical chemical descriptors versus IPPSF for pharmacokinetic parameter "b”.
  • Figure 13 is a plot of physical chemical descriptors versus IPPSF for pharmacokinetic parameter "d”.
  • Figure 14 illustrates an exemplary general purpose computing platform 100 upon which the methods and systems of the present invention can be implemented.
  • Figure 15 is a schematic perspective view of one embodiment 50 of a membrane assembly.
  • a skin-imitating membrane is coated on a section of inert fiber to be used as a permeation membrane.
  • the membrane-coated fiber (MCF) is immersed in the donor phase to partition the compounds into the membrane.
  • MCF membrane-coated fiber
  • the membrane- coated fiber is transferred into the injection port of a gas chromatograph or a high-performance liquid chromatograph to desorb the compounds for quantitative and qualitative analyses.
  • Many compounds can be studied at a single run because of the high separation power of the chromatographic techniques. This feature is useful to study the synergistic effect of multiple chemicals and their combinations on percutaneous absorption. Expensive radiolabeled compounds are not required.
  • This membrane-coated fiber characterizes a half compartment of the conventional diffusion chamber, which allows more detailed permeation kinetics to be investigated.
  • a theoretical model is provided and describes the permeation processes of the skin-imitating membrane coated fiber.
  • An absorption container is designed to incorporate the membrane-coated fiber to meet the requirements of the theoretical derivation for percutaneous absorption.
  • physical chemical interactions, characteristics or factors that significantly alter in vivo dermal absorption after exposure in a complex chemical mixture are primarily related to solute/solvent, solute/solute, solvent/membrane or solute/membrane interactions that are detectable in an appropriately optimized and parameterized in vitro system.
  • the present invention provides an experimental approach that defines these interactions in terms of molecular descriptors that make the results applicable to other pharmaceutical and toxicological problems.
  • the MCF approach is employed to experimentally measure multiple partition coefficients (log Km/s) between study chemicals and a number of physical and chemically diverse membranes (m) in biologically relevant solvent(s) systems.
  • the log K m/S is scaled to the molecular descriptors by specific intermolecular forces defined by a linear solvation energy equation.
  • These molecular descriptors also parameterize the IPPSF model, providing the link between the MCF and IPPSF systems.
  • Multiple MCFs are calibrated using compounds with known values of the molecular descriptors with multiple solvation energy equations describing log K m/S . These calibrated MCFs are then used as a reference system to determine the molecular descriptors for any study chemicals of toxicological significance.
  • log K m/S are determined in the mixture or solvent solutions using the calibrated MCFs. Change in a chemical's log K m/S relative to the reference system's, reflective of mixture effects, is quantitated as apparent change ( ⁇ ) in a chemical's molecular descriptors from reference control values. This provides an experimental approach for quantitative assessment of chemical mixture exposure scenarios. Since the IPPSF's in vivo absorption estimates are parameterized in terms of the molecular descriptors, ⁇ values can be linked to the IPPSF to predict changes in absorption profiles.
  • a strength of the IPPSF model is its sensitivity to biological effects.
  • changes in IPPSF flux profiles due to biological modifiers can be assessed using independent markers of biological activity (e.g. inflammatory cytokine IL-8 release, changes in infrared spectra in stratum corneum determined using FTIR) and then integrated into the model as explanatory or concomitant variables.
  • independent markers of biological activity e.g. inflammatory cytokine IL-8 release, changes in infrared spectra in stratum corneum determined using FTIR
  • This approach allows a direct identification of mixture or solvent effects that would be expected to significantly alter in vivo disposition.
  • L Candidate Compounds The term “candidate compound” is meant to refer to any compound wherein characterization of the compound's susceptibility to percutaneous absorption is desirable.
  • Exemplary candidate compounds include xenobiotics such as drugs and other therapeutic agents; carcinogens and environmental pollutants; pesticides; and endobiotics such as steroids, fatty acids and prostaglandins.
  • xenobiotics such as drugs and other therapeutic agents
  • carcinogens and environmental pollutants such as insects and other therapeutic agents
  • pesticides such as benzyl alcohol
  • endobiotics such as steroids, fatty acids and prostaglandins.
  • Other representative candidate compounds, including those employed for calibration, are disclosed in the Examples.
  • the terms “solute”, “penetrant”, and/or “solute/penetrant” can be used herein interchangeably with the term “candidate compound”.
  • candidate compounds screened in accordance with the method of the present invention are contemplated to be useful in the treatment of warm-blooded vertebrates. Therefore, the invention concerns mammals and birds.
  • domesticated fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans.
  • livestock including, but not limited to, domesticated swine (pigs), ruminants, horses, poultry, and the like.
  • QSPR quantitative structure permeability relationships
  • El Tavar et al. (1991) analyzed subsets of compounds from the Flynn dataset and described a correlation with a hybrid parameter ⁇ log K oc t- h ep (Koct/ ater- Kheptane/water), which provides an estimate of the hydrogen bond donor acidity of the solutes.
  • Puqh and Hadgraft (1994) analyzed this same data set using an ab initio approach using up to 17 fragments based on various molecular substructures and features and achieved a comparable correlation to Potts and Guy. This study identified a number of outlier compounds that were also identified by other workers subsequently analyzing this dataset.
  • the present invention quantifies the relationship between experimentally determined partition coefficients and a chemical's molecular descriptors using the MCF technique to measure a series of partition coefficients in different membranes of diverse physical chemical properties.
  • the isolated perfused porcine skin flap system (IPPSF) described herein provides estimates of dermal absorption.
  • the IPPSF model has been shown to correlate to human in vivo absorption and has been shown to be sensitive to biological modifiers such as chemical induced irritation or vascular activity. Once the MCF systems are calibrated, the effects of a specific chemical mixture component can be tested in these systems.
  • the MCF technique is an experimental system closer to the basic properties embodied in a theoretical QSPR analysis.
  • the IPPSF moves the in vitro skin model closer to the biological functionality inherent to in vivo studies, without the presence of confounding systemic factors and procedural or ethical hurdles inherent to conducting both animal and human trials.
  • Linear solvation energy relationships can be used to predict skin absorption and to make observations that can be generalized across molecules of very different physical chemical properties, as well as levels of model systems.
  • QSPR studies have defined a set of molecular descriptors, including, for example, descriptors that related to hydrogen bonding. It is well known that hydrophobicity of a compound plays a very important part in partitioning, permeation, and deposition in a biological system. Molecular volume, often roughly estimated as molecular weight in early analyses, is also an important geometric factor of the compound. In Abraham's linear solvation energy relationship, V x and logL were used alternatively for solution-membrane and gas-membrane systems (Eq.3).
  • Intrinsic molecular volume is used for the molecular volume term that can be calculated for any compound, such as by using the computer program MOLSV (QCPE, Indiana).
  • the hydrophobicity term is the partition coefficient of a chemical between gas phase and a hydrophobic MCF (without polar type or H-bonding capacity) that is expected to be similar to the gas-hexadecane partition coefficient available in the art.
  • the variables [R, ⁇ , , ⁇ , V and L] are molecular descriptors of the solute. Each of the solute descriptors represents the strength of the corresponding intermolecular force of the solute. The values of these descriptors are available in the art for the calibration compounds disclosed in the Examples below.
  • R is an excess molar refraction that can be calculated from refractive index.
  • ⁇ is the effective solute H-bond acidity, a summation of acidity from all H-bonds of the solute.
  • ⁇ is the effective solute H-bond basicity, a summation of basicity from all H-bonds of the solute.
  • V is the intrinsic volume of solute that can be calculated (such as by using the MOLSV software package)
  • L is the solute gas-hexadecane partition coefficient at 25°C.
  • the constants [c, r, s, a, b, v and I] are parameters of the membrane/solvent system for which log Km /S is being measured. Each of the system parameters represents the strength of the corresponding intermolecular force of the system. These strength coefficients essentially scale the contribution of each molecular descriptor in determining the log K m/S to the specific fiber being studied. These system constants can be obtained through data regression of specifically designed experimental measurement by the
  • the r-constant shows the tendency of the system to interact with solutes through ⁇ * - and n-electron pairs. Usually the r-coefficient is positive, but for phases that contain fluorine atoms, the r-coefficient can be negative.
  • the a-constant denotes the hydrogen-bond basicity of the system (acidic solutes will interact with a basic membrane).
  • the b-constant is a measure of the hydrogen-bond acidity of the system (basic solutes will interact with an acidic membrane).
  • the v-constant is a measure of the endoergic cavity term of the system excluding any form of intermolecular interactions.
  • the l-constant is a combination of exoergic dispersion forces that make a positive contribution to the l-coefficient. It mainly measures the hydrophobicity of the system. Aspects of the above-listed molecular descriptors have been studied for three decades for solvent and solute properties, and appear to be the most general set of parameters currently available, as well as the set that would offer the best extrapolations to other work in the art. An advantage of describing skin permeability relationships using such descriptors is that information is also gained on the mechanism of permeation. In the skin chemical mixture experiments conducted in the art to date, dermal absorption is empirically assessed as epidermal permeability (steady state parameter) or flux through skin (e.g.
  • the isolated perfused porcine skin flap (IPPSF) system is a single-pedicle, axial pattern tubed skin flap obtained from the abdomen of female weanling Yorkshire pigs (Sus scrofa). Two flaps per animal, each lateral to the ventral midline, are created in a single surgical procedure. As depicted in Figure 8, the procedure involves surgical creation of the flap (measuring 4 cm x 12 cm) perfused primarily by the caudal superficial epigastric artery and its associated paired venae comitantes (Step A & B), followed by arterial cannulation and harvest in 48 hours (Step C) (Bowman et aL, 1991).
  • the IPPSF is then transferred to a perfusion apparatus that is a custom designed temperature and humidity regulated chamber (Figure 9).
  • the media comprises a modified Krebbs Ringer buffer with bovine serum albumin. Normal perfusate flow is maintained at 1 ml/min/flap (3-7 ml/min/100g) with a mean arterial pressure ranging from 30-70 mm Hg, targets consistent with in vivo values reported in the art. Viability for up to 24 hours has been confirmed through biochemical studies and extensive light and transmission electron microscopy studies (Monteiro-Riviere et al., 1987).
  • Compounds can be topically applied neat or diluted in vehicle under ambient (non-occluded) or occluded conditions.
  • a relatively large dosing area of up to 10 cm 2 is available for compound application and is an advantage of this system. This allows for drug delivery patches, iontophoretic devices, and an applied surface area large enough to be comparable to human application.
  • IPPSF venous efflux profiles have been analyzed using a number of pharmacokinetic models (Williams et al., 1990; Williams and Riviere, 1995). These models integrate basic Fickian diffusion parameters to pharmacokinetic parameters based on defining differential equations. IPPSFs are the most complex model employed in these studies since it has been shown to be predictive of in vivo human absorption under a variety of exposure scenarios (Riviere et al.. 1992, 1995, 2002, 2003; Wester et al., 1998). These data are plotted in Figure 10 for 16 compounds where comparable experimental conditions (dose/unit area; vehicle) for both data sets were available.
  • Pharmacokinetic modeling allows integration of relevant physical chemical parameters into a model predictive of IPPSF (and by extension in vivo human) absorption. It is based on defining IPPSF absorption profiles using the simplest pharmacokinetic model that reflects rate-limiting processes involved in absorption without relying on complex mathematically non-identifiable compartmental models previously used (Williams et al., 1996). This simplified model describing flux (Y(t)) at time t is:
  • IPPSF flux profiles can be characterized by five physical chemical parameters: H acidity, H basicity, V m , S-Polarizability and H 2 O solubility.
  • Figure 3 shows an embodiment of a simulated biological membrane (e.g. a skin-imitating membrane) coated fiber (MCF).
  • a piercing needle 4 is attached to a needle base 3.
  • a sealing septum 2 is inserted in the needle base 3b.
  • the needle base has a taper end 3c for positioning during absorption.
  • Fiber attachment tubing 5 can slide inside of the piercing needle through the sealing septum.
  • the top end of the fiber attachment tubing is attached to a holding tip 1.
  • a chemically inert fiber 6 is attached to the lower end of the fiber attachment tubing.
  • One section of the inert fiber is coated with a membrane 7.
  • This simulated biological membrane is used to partition compounds from solutions, and desorb the partitioned compounds into the injector of a gas chromatograph (GC) or a high performance liquid chromatograph (HPLC) while keeping the membrane unchanged.
  • the membrane has characteristics that cover a wide-strength range of molecular interactions. Indeed in one embodiment, as large a range as possible is provided.
  • the membrane can simulate a biological membrane (as defined herein) of interest.
  • a skin-imitating membrane preferably has similar absorption properties as skin.
  • the membrane preferably has high thermal stability for desorption of the compounds into the GC injector without damage to the membrane itself; or have high solvent stability to desorb the compounds into the HPLC column without damage to the membrane itself.
  • PDMS polydimethylsiloxane
  • polyacrylate polyacrylate
  • crosslinked PDMS polydimethylsiloxane-polycarbonate block copolymer
  • other stationary phases used in GC columns and HPLC columns that provide similar absorption properties as skin. Additional representative materials are disclosed in the Examples and indeed, in one embodiment of the present invention, the use of multiple materials, alone and in combination, is provided.
  • the exposing surface area (A) of the skin-imitating membrane is determined by the membrane coating radius (R m ) and the coating height (H).
  • the volume V m of the skin-imitating membrane is determined by the membrane thickness ( ⁇ m ), the coating radius (R m ) and the coating height (H).
  • the volume (V d ) is the volume of the donor solution.
  • the holding tip 1 and the needle base 3 are made of plastic material.
  • the piercing needle 4 and the fiber attachment tubing 5 are made of stainless steel.
  • the inside of the fiber attachment tubing 5 is filled with reinforcing material to increase its mechanical strength.
  • the inert fiber 6 is a fused-silica fiber or a stainless steel fiber, or other suitable inert fiber as would be apparent to one of ordinary skill in the art after a review of the disclosure of the present invention set forth herein.
  • Figure 4 shows the design of the absorption container with a special needle holding cap and water jacket.
  • the needle holding cap 10 has several holes drilled in a specific shape 12 to fit the taper end of the needle base 3. The number of holes is determined by the number of needle to be used, for instance, 4, 6, 8 or more.
  • All of the holes 12 are on the same radius R c , which is the radius of the membrane coated fiber to the vertical centerline of the container.
  • the needle holding cap 10 is well fit into the solution container 20 for precise control of the radius R c .
  • the membrane-coated fiber is immersed in the donor solution 26 for partitioning the compounds from the solution.
  • the temperature of the solution is maintained by the water jacket 22 with inlet 25 and outlet 24.
  • the needle holding cap 10 is made of plastic materials by molding or machine.
  • the absorption container 20 is preferably made of glass or TEFLON® PTFE to reduce its chemical absorption.
  • FIG. 5 shows the experiment setup for percutaneous absorption study with the membrane-coated fiber.
  • the absorption container 20 sits on a magnetic stirrer 30.
  • a magnetic stir bar 21 is stirring the solution 26 in the absorption container.
  • the stirring rate is controlled by a rate controller 34 and displayed on a tachometer 32.
  • Needle holding cap 10 positions the membrane-coated fibers 3.
  • all of the geometric parameters, such as, R c , membrane coating radius R, membrane thickness ⁇ m are preferably kept constant (i.e. uniform) with the present setup.
  • the water jacket maintains the solution at constant temperature.
  • Membrane assembly 50 comprises a membrane 52 mounted inside tube 54.
  • One, two, or indeed any desired number of membranes 52 can be mounted inside tube 54, as is feasible based on the dimensions of membrane 52 and tube 54.
  • Membranes 52 can be the same or different.
  • Tube 54 can comprise a stainless needle (e.g. stainless steel), capillary tube, cylinder, or another suitable structure as would be apparent to one of ordinary skill in the art after a review of the present disclosure. Additionally, tube 52 can have any suitable cross-sectional configuration, such as but not limited to one of circular, triangular, and rectangular (including a square). Assembly 50 allows compounds to partition into membrane 52 inside tube 54 and to desorb into the GC injector for quantitative analysis.
  • the present invention can be implemented in hardware, firmware, software, or any combination thereof.
  • the methods and data structures for determining a molecular descriptor of absorption for a candidate compound and/or for assessing susceptibility of a candidate compound to absorption into a biological membrane can be implemented as computer readable instructions and data structures embodied in a computer-readable medium.
  • the data structures can comprise one or more data fields (e.g. 2,3,4,5 or more such data fields) containing molecular descriptors of different types and values, e.g. reference molecular descriptors.
  • Computer readable instructions for implementing Equations 7, 8, and 10 are also provided.
  • an exemplary system for implementing the invention includes a general purpose computing device in the form of a conventional personal computer 100, including a processing unit 101 , a system memory 102, and a system bus 103 that couples various system components including the system memory to the processing unit 101.
  • System bus 103 can be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures.
  • the system memory includes read only memory (ROM) 104 and random access memory (RAM) 105.
  • ROM read only memory
  • RAM random access memory
  • a basic input/output system (BIOS) 106 containing the basic routines that help to transfer information between elements within personal computer 100, such as during start-up, is stored in ROM 104.
  • Personal computer 100 further includes a hard disk drive 107 for reading from and writing to a hard disk (not shown), a magnetic disk drive 108 for reading from or writing to a removable magnetic disk 109, and an optical disk drive 110 for reading from or writing to a removable optical disk 111 such as a CD ROM or other optical media.
  • Hard disk drive 107, magnetic disk drive 108, and optical disk drive 110 are connected to system bus 103 by a hard disk drive interface 112, a magnetic disk drive interface 113, and an optical disk drive interface 114, respectively.
  • the drives and their associated computer-readable media provide nonvolatile storage of computer readable instructions, data structures, program modules, and other data for personal computer 100.
  • a number of program modules can be stored on the hard disk, magnetic disk 109, optical disk 111, ROM 104, or RAM 105, including an operating system 115, one or more applications programs 116, other program modules 117, and program data 118.
  • System memory 104 and/or 105 can also include a search engine, a database manager, and a comparator program having instructions for implementing the search, management, compilation (e.g. addition and deletion of molecular descriptors from database or other aspects of memory), comparing data, assessing data, and displaying the molecular descriptors of absorption for a candidate compound and comparisons thereof.
  • search engine and database manager can include a software database application such as FILEMAKER 5.5v2 UNLIMITED produced by FileMaker, Inc. of Santa Clara, California, United States of America. Other software programs and packages are disclosed in the Examples.
  • a user can enter commands and information into personal computer 100 through input devices such as a keyboard 120 and a pointing device 122.
  • Other input devices can include a gas chromatograph, a high performance liquid chromatography apparatus, a microphone, touch panel, joystick, game pad, satellite dish, scanner, or the like.
  • serial port interface 126 that is coupled to the system bus, but can be connected by other interfaces, such as a parallel port, game port or a universal serial bus (USB).
  • a monitor 127 or other type of display device is also connected to system bus 103 via an interface, such as a video adapter 128.
  • personal computers typically include other peripheral output devices, not shown, such as speakers and printers.
  • the user can use one of the input devices to input data indicating the user's preference between alternatives presented to the user via monitor 127.
  • Personal computer 100 can operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 129.
  • Remote computer 129 can be another personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to personal computer 100, although only a memory storage device 130 has been illustrated in Figure 14.
  • the logical connections depicted in Figure 14 include a local area network (LAN) 131 , a wide area network (WAN) 132, and a system area network (SAN) 133.
  • LAN local area network
  • WAN wide area network
  • SAN system area network
  • System area networking environments are used to interconnect nodes within a distributed computing system, such as a cluster.
  • personal computer 100 can comprise a first node in a cluster and remote computer 129 can comprise a second node in the cluster.
  • remote computer 129 it is preferable that personal computer 100 and remote computer 129 be under a common administrative domain.
  • computer 129 is labeled "remote"
  • computer 129 can be in close physical proximity to personal computer 100.
  • personal computer 100 When used in a WAN networking environment, personal computer 100 typically includes a modem 138 or other device for establishing communications over WAN 132. Modem 138, which can be internal or external, is connected to system bus 103 via serial port interface 126. In a networked environment, program modules depicted relative to personal computer 100, or portions thereof, can be stored in the remote memory storage device. It will be appreciated that the network connections shown are exemplary and other approaches to establishing a communications link between the computers can be used.
  • the present invention pertains to percutaneous absorption, it is further provided that other biological barriers and membranes can be analyzed.
  • Representative barriers and/or membranes include but are not limited to subcellular, cellular, oral/mucosal, gastrointestinal, blood-brain, respiratory-lung, nasal, ocular and subconjuctival.
  • simulated biological membrane is meant to encompass other biological barriers and membranes, including but not limited to those listed above, as well as the skin-imitating membrane disclosed above.
  • the materials disclosed above and in the Examples that can be used alone or in combination to prepare the skin-imitating embodiment of a membrane of the present invention can also be used alone or in combination to prepare a simulated biological membrane of the present invention.
  • the membrane has characteristics that cover a wide-strength range of molecular interactions. Indeed in one embodiment, as large a range as possible is provided.
  • This complicated absorption mechanism can be separated into two i sections, permeation section and penetration section.
  • the permeation section comprises the donor phase, the boundary layer ( ⁇ d ), and the membrane;
  • the penetration section comprises the membrane, the boundary layer ( ⁇ r ), and the receptor phase.
  • Fick's first law of diffusion can be expressed as follows for a continuous flowing system at the boundary region between the donor phase and the skin-imitating membrane:
  • F the diffusion flux of a given permeant from the donor phase to the membrane surface, which is equal to the diffusion flux of the permeant from the membrane surface into its inner membrane phase for a balanced mass transfer
  • A surface area of the silastic membrane
  • D d is the diffusion coefficient of the permeant in the donor phase
  • D m is the diffusion coefficient of the permeant in the membrane phase
  • Ca and C m are concentrations of the permeant in donor phase and in membrane phase, respectively
  • x is an axial perpendicular to the membrane surface.
  • the diffusion flux can also be expressed in permeation time as follows:
  • C d is the concentration of the permeant in the bulk donor phase.
  • Cdx ' is the surface concentration of the permeant in the donor phase, which is the driving force for the permeant diffused into the membrane
  • ⁇ d is the thickness of the donor boundary layer
  • Cm d is the concentration of the permeant in the membrane surface
  • C mr is the concentration of the permeant in the membrane contacting the fused silica fiber
  • ⁇ m is the thickness of the skin-imitating membrane as shown in Figure 3.
  • the partition coefficient is expressed as follows:
  • the concentration gradient of the permeant can be approximated to be linear in the membrane phase. Then, the permeation amount can be approximated by the mean concentration increase as follows:
  • the first item ( ⁇ n/D m ) is the contribution of the membrane while the second item (2K ⁇ /Dd ) is the contribution of the boundary layer.
  • the partition coefficient ( ) is large enough to satisfy a condition, ⁇ n /Dm «2K ⁇ d/Dd , the contribution of the membrane can be neglected: (MFC _ 1 8)
  • the diffusion coefficient in the membrane (D m ) can be calculated from the measured parameters by rearranging Eq.MFC-11 :
  • FIG. 6 shows a GC/MS spectra acquired with a HP 5890 gas chromatograph coupled with a HP 5970B mass selective detector and a polydimethylsiloxane (PDMS) membrane coated fiber (MCF) partitioned for 30 minutes in a solution containing 30 compounds.
  • the injection port was maintained at 280 9 C for sample vaporization and thermal desorption. Separation was performed on a 30 m x 0.25 mm (i.d.) x 0.25 ⁇ m (df) Rtx-5MS capillary column (Restek Corp., Bellefonte, Pennsylvania, United States of America).
  • the transfer line temperature was set at 250 Q C.
  • the column oven was programmed as follows: held at the initial temperature 100 9 C for 1 min., ramped at 15 9 C/min to 150 9 C, 1 9 C/min to 220 e C and 3 9 C/min. to 280 9 C, and held for 5 min.
  • An electronic pressure control was used to maintain a carrier gas flow of 1.00 mL/min helium.
  • the membrane-coated fiber was conditioned in flowing helium at 280 9 C for 10 min.
  • the absorption experiments were performed as follows.
  • the membrane-coated fiber was positioned in the absorption container and the membrane section was immersed into the solution to be studied.
  • the solution was a standard mixture containing 30 components having a wide range of octanol-water partition coefficients. The concentration of each compound in the solution is 8 ng/mL.
  • the solution was stirred at 400 rpm. After the membrane-coated fiber was immersed in the solution for a given time, the membrane-coated fiber was transferred directly into the injection port of the gas-chromatograph for quantitative analysis.
  • Figure 7 shows the absorption amount versus time profiles for three compounds, Dacthal, Heptachlor Epoxide and tr-Nonachlor. The experimental procedures are same as in Figure 6. The absorption time was set as 5, 15, 30,
  • Vm n° in the donor phase (C d e - C° ) is governed by the partition equilibrium:
  • Eq. MCF-21 is exactly the same form as Eq. MCF-15a derived from the proposed theoretical model. This establishes that the theoretical derivation and assumptions made are adequate.
  • the partition coefficient " can be calculated from Eq. MCF-15a if the equilibrium amount (n°) is known.
  • there are at least two optional methods to obtain the equilibrium permeation amount One is to measure the permeation amount at prolonged equilibration time, at which equilibrium permeation is assumed to be reached. This is easy for small compounds with lower partition coefficients. For larger compounds with high partition coefficients equilibrium cannot be reached for days or weeks.
  • Another method is to use the initial permeation amounts sampled in a limited period of time, and use the Eq. MCF-16 to simulate the equilibrium amount as described in the following.
  • Dd A ⁇ ⁇ MCF - 22 >
  • D d is the diffusion coefficient of a given compound in the donor solution (cm 2 /s)
  • V a is the molar volume of the compound (cm 3 /mole).
  • the molar volumes of the compounds were compiled from published reference database.
  • the thickness ( ⁇ m), length (h) and volume (V m ) of the membrane-coated fiber were 100 ⁇ m, 1.00 cm and 0.612 ⁇ l, respectively.
  • the thickness of the boundary layer ( ⁇ d ) was estimated by an approaching method, i.e., ⁇ d value was calculated for each compound with Eq. MCF-18a from the obtained parameters ( a and K) and the estimated D d value.
  • Examples 3 and 4 Experimental Determination of Molecular Descriptors
  • the focus of these Examples is to adapt the general solvation equation for partition coefficients to allow for experimental determination of the molecular descriptors for any study compounds using the MCF technique in a framework coupled both to predicting percutaneous absorption in the IPPSF (which is equivalent to predicting absorption in vivo, such as in human beings) and assessing chemical mixture or solvent effects.
  • the linear solvation energy relationship (Eq.4) is the basis for linking these physical chemical properties of a penetrant to permeation through skin using the IPPSF model. Similar molecular descriptors appear to suitably predict disposition of a topically applied compound in the IPPSF.
  • the focus of these experiments is to develop an experimental approach using the MCF technique to calculate the molecular descriptors [R, ⁇ , ⁇ , ⁇ , and L] suitable for prediction of [A, b and d] for a specific penetrant as parameterized in the IPPSF kinetic model.
  • These parameters are experimentally determined in MCFs so that effects of solvent and chemical interactions in subsequent experiments can be quantitated based on changes in a fiber's log Km / s mapped onto values of the molecular descriptors that link both the MCF and IPPSF systems.
  • a series of calibration compounds are used to estimate the system parameters for a specific fiber in the water solvent system.
  • This approach employs the MCF technique to generate descriptors in an aqueous system at 37°C for subsequent use in evaluating mixture chemical interactions or solvent effects.
  • Different fibers are selected to model specific types of interactions.
  • a polydimethylsiloxane (PDMS, silicone) MCF fiber was used to estimate K oct anoi/water-
  • PDMS polydimethylsiloxane
  • silicone is biased toward hydrophobic interactions and could only be sensitive to mixture effects involving these classes of skin constituents. Additional partition coefficients are utilized to obtain better in vivo predictions as well as to detect more subtle chemical mixture or solvent interactions that can be biologically relevant to altered dermal absorption.
  • Polysiloxane and polyethylene glycol (PEG) polymers are the primary stationary phases used in modern gas chromatography. These two polymers can be crosslinked and uniformly coated onto the surfaces of fused-silica fibers or inside of the capillary columns. The physiochemical properties of these two polymers can be easily modified by integrating different functional groups into the polymer backbones.
  • the membrane-coated fibers can be prepared using any preparation technique that is generally suitable for preparing a stationary phase in gas chromatography, as would be apparent to one of ordinary skill in the art after a review of the present disclosure.
  • Two representative methods are as follows. The first method is to coat the membrane on a section of fused-silica fiber as used in Examples 1 and 2.
  • Four MCFs (#2, 13-15 in Table II) of this type are commercially available for use in solid-phase extractions.
  • the PEG polymer based MCFs in Table III are prepared with a sol-gel technology developed by Wang et al. (2000).
  • the functionally modified PEG backbone with end- hydroxyl groups is integrated into the sol-gel matrix to form a thermal and chemical stable cross-linked membrane.
  • the polysiloxane based MCFs (#3-#8 in Table II) are prepared with procedures developed by Lee and coworkers (Peaden at al. 1982; Kong et al. 1984).
  • the polysiloxane polymer with given ratios of functional groups and 1% vinyl-polysiloxane is coated on to an inert fiber (fused-silica or stainless steel), then the polymer is crosslinked by the vinyl groups initiated by radicals.
  • PSF6 (#1 in Table II) is a hydrogen-bond acid stationary phase developed by Martin, Pool et al. (1998), in which the polymethylsiloxane backbone comprises 4-butanephenyl and hexafluoropropanol-2.
  • the second method employs a section of the GC capillary column and mounts it inside a stainless needle, which is modified to allow compounds partitioning into the membrane inside the capillary tube and desorbing into the GC injector for quantitative analysis.
  • Such capillary columns are commercially available for all of the membrane materials in Table II except #1.
  • Calibration Compounds Fifty calibration compounds with known molecular descriptors of varying physical chemical properties are selected (Table III). Some of the molecules are chosen to have extreme values (e.g.0) in the descriptor matrix, which significantly increases the statistical power of the study. These compounds were selected as being amenable to GC/MS analysis and have known molecular descriptor values.
  • [R, ⁇ , , ⁇ , L and V] are the estimated parameters.
  • the purpose of estimating these descriptors is to generate values for parameterizing the IPPSF model and providing reference descriptors in water as a solvent to subsequently detect chemical mixture or solvent interactions. It is assumed that such interactions would not be reflected in changes in the intrinsic molecular volume
  • Equation 8 Equation 8 above thus reduces to:
  • the compounds selected to be parameterized are those that are also used to study percutaneous absorption in additional MCF and IPPSF experiments.
  • Table IV lists the 40 chemicals selected for this purpose.
  • Methyl nicotinate is a known vasodilator. These compounds are included as their rate and extent of percutaneous absorption could be strongly influenced by their activity in skin.
  • each chemical should be studied in 15 fibers as formulated above.
  • the complexity of the experimental system is reduced to one of more manageable proportions when used in the chemical and solvent interaction Examples.
  • the above system calibration matrix is statistically analyzed using step-wise subtraction of fiber vectors as well as a principal component analysis from the regression matrix to determine the minimal number of fibers that contribute adequate predictability to determine the five system parameters.
  • a maximum of eight fibers are selected to determine the molecular descriptors of the study chemicals. These eight fibers are then used as a base experimental system for MCF studies involving solvent and mixture effects.
  • results of this correlation analysis could indicate that one or more of the above molecular descriptors [R, ⁇ , , ⁇ , and L] is redundant (e.g. highly correlated to a different parameter) and does not offer significant statistical power in determining fiber partition coefficients.
  • a process similar to the formulation of the Z parameter is conducted to collapse the variable into constant Z. This is addressed by step-wise regression techniques, which reduce the number of parameters estimated in subsequent phases of this Example.
  • Example 2 provides a method to quantitatively assess the mixture and vehicle effects using the solute descriptors obtained from the MCF technique.
  • the eight fibers selected in Example 3-2 above cover a wide strength range of intermolecular interactions.
  • the system constants are determined for each fiber in Example 3-1 in water, where no solute-solute intermolecular interactions exist outside of the solute-membrane and solute-water interactions.
  • the ⁇ parameters reflect the altered strength coefficients ( ⁇ ) for each descriptor secondary to the mixture or solvent effect, and are used here to facilitate discussion of these interactions independent of the MCF experimental context that generated them. This distinction is a tool to aid in their subsequent interpretation in the mixture and solvent experiments, as well as in the final biological IPPSF absorption model.
  • the apparent molecular descriptors [R', ⁇ ', ', ⁇ ', U] correlate to solvation energy related physiochemical properties (e.g. partition coefficient, permeation constant; solubility) since they reflect all of the intermolecular interactions in the mixture.
  • the apparent descriptors are obtained in the MCF interaction experiments. In this framework, these apparent descriptors are used to parameterize [A, b, d] in the IPPSF model after mixture or solvent exposures.
  • Solvent Effects Five solvent systems are employed to probe potential interactions compared to the water reference system. These include 50% water/ 50% ethanol (the second solvent systems used in the IPPSF exposures) as well as 100% ethanol; 100% acetone; 100% cyclohexane; and 100% propylene glycol. The selection of test solvents is based in part on two different criteria: (1) relevance to dermal absorption; and (2) chemical property dissimilarity to "force" significant descriptor interactions to occur. Ethanol, aqueous ethanol and acetone were selected because similar solvents have been often used in human and laboratory animal studies (Cross et al., 2001 ; Zatz, 1993), including mixture interaction experiments that demonstrated significant modulation of in vivo absorption (see e.g.
  • Example 3-2 Cyclohexane has also been employed as a solvent in topical exposures (King and Monteiro-Riviere, 1991 ) and provides a different physical chemical environment to test the solute descriptors.
  • the 20 compounds selected in Example 3-2 above are studied for a total of 20 chemicals x 5 solvents or 100 experiments conducted in all eight fibers with 3-9 replicates, for reasons similar to that discussed in Example 3-2.
  • This phase generates a series of ( ⁇ R, ⁇ , ⁇ , ⁇ , ⁇ L) SO ivent which can be evaluated to determine those solvent/solute combinations which have the largest effects on absorption.
  • a number of binary and multi-component mixtures (2, 4, 8 12, 16 and 20 components) are created from the 20 compounds selected above for IPPSF studies. Doses of mixture components are titrated to produce a significant ⁇ for the chemical being monitored. Those mixtures providing the largest ( ⁇ R, ⁇ , ⁇ , ⁇ , ⁇ L) m ixture values are selected for IPPSF studies described in Example 4 below. This is a strength of the MCF fiber technique as the IPPSF studies probe the values of ⁇ that significantly change percutaneous absorption. Additionally, since this approach links the solvent/solute mixture interactions to specific molecular descriptors, mechanisms of interactions that significantly alter absorption can be described.
  • mixtures are composed from the set of 20 chemicals selected in Example 3-2. Eighty experimental mixtures are randomly determined and spread over 2, 4, 8 12, 16 and 20 (total mixture) combinations.
  • Mixed effects modeling is used to identify and characterize factors that alter the cutaneous absorption of toxic substances in chemical mixtures. This modeling technique makes it possible to identify sources of variability, particularly if there is a broad range of factors of potential relevance with unknown relationships present. Physical chemical interactions that alter solvation properties, detected in MCF studies, as well as biologically based interactions are expected to affect the absorption of chemicals in the IPPSF. Mixed effects modeling can be used as a tool to identify and characterize these interactions.
  • Perfusate flux-time data (Y(t)) obtained from the IPPSF experiments is fitted to the previously defined bi-exponential structural model (Eq. 5) using non-linear regression:
  • Parameter A relates to the amount of applied dose ultimately absorbed
  • parameter b reflects the rate of terminal release of the absorbed chemical
  • parameter d reflects the rate of uptake of the chemical from the skin surface.
  • Mixed-effect modeling has been widely applied in human clinical pharmacology for decades (Sheinerand Beal, 1980, 1981 , 1983) and is now embodied in the commercially available software package marketed under the trademark WINNONMIX ® (Pharsight Corporation, Cary, North Carolina, United States of America).
  • the predicted values for the flux of a chemical compound differ from measured values. In a mixed effects model, these differences are attributed to both fixed and random effects. Predictions should be improved if parameters A, b and d are calculated more accurately by accounting for a number of fixed effects that reflect both physico-chemical and biologically based interactions. This can be done by considering both solute descriptors [R', ⁇ ', ⁇ ', ⁇ ', V and log L'] as well as indicators of biological activity (IL8 release from keratinocyte cell cultures and shifts in FTIR stratum corneum spectra) as co-variates in the mixed effect model, in the following manner:
  • Aavg ⁇ i + ⁇ 2 R' + ⁇ 3 ⁇ ' + ⁇ 4 ⁇ ' + ⁇ 5 ⁇ ' + ⁇ 6 V + ⁇ 7 logL' + ⁇ 8 ( ⁇ IL8 release) +
  • This model is fit using the 20 study compounds selected from Example 3-2 dosed in both water and ethanol/water vehicles.
  • the apparent descriptors [R', ⁇ ', ⁇ ', ⁇ ', L'] and V, which reflect the potential ethanol effects, are used to establish the ⁇ correlation matrix, ⁇ is independent of any solvent effects, as this information is embedded in the apparent descriptors. Lack of fit is reflected in ⁇ .
  • This design yields 40 IPPSF treatments replicated 4 times (160 IPPSFs).
  • Nonlinear mixed effects modeling are performed using the commercially available software program WINNONMIX ® (Pharsight Corporation). This program offers a number of different algorithms and regression techniques that can be used to estimate parameters and their variability.
  • the first step involves specifying the structural pharmacokinetic model (in this case, the bi-exponential model described above).
  • the program has the capability to consider a total of 36 different fixed effects parameters to improve the estimation of population pharmacokinetic parameters.
  • the relationship of these fixed effects parameters to the pharmacokinetic parameters can be described by means of linear, multiplicative, exponential and logarithmic equations. "Goodness of fit" is reflected in a statistically significant decrease in the minimum value of the objective function (MVOF).
  • AIC Akaike's Information Criterion
  • SBC Schwarz's Bayesian Criterion
  • Model building is approached in an iterative manner by adding/changing fixed effects parameters and determining how this improves/weakens the model. Normal distribution is assumed and parametric techniques are used to calculate variances. Ideally, identifying and correctly characterizing as many covariates as possible and accounting for these in the fixed effects minimize random effects. As modeling proceeds, if the values of the variance ( ⁇ ) are large for specific chemicals, this would suggest another factor or biological modifier is present (e.g. vasoactivity) that might have to be included as an indicator (+/-) variable. All perfusate samples are analyzed using GC/MS that allows for detection of metabolites. As discussed herein above, compounds must first pass the stratum corneum before they are accessible for drug metabolism. In these scenarios, total penetrating activity driving diffusion (parent drug and metabolite) is used in the analysis, and any deviation from predicted concentrations is allocated to metabolism.
  • AIC Akaike's Information Criterio
  • IPPSF Experimental Designs Studies are conducted occluded with ethanol and ethanol-water dosing solutions applied at a concentration of 40 ⁇ g/cm 2 to a 5 cm 2 surface area. The two vehicles provide altered absorbed dose information for the mixed effect model. These conditions also simulate fiber exposures as well as provide a link to existing IPPSF data and to other in vitro and in vivo exposures in the art. Perfusate samples are collected for 8 hours (0, 10, 20, 30, 456075 and 90 min, and every Vz h until termination at 8 h). All samples are analyzed for compound absorption using GC/MS. Treatments are repeated in 4 flaps, and this number has been shown to be an efficient number for statistical comparisons. This allows for calculation of a variance for the error term in the mixed effects model described below.
  • Example 4-1 Example 4-1
  • IPPSF absorption studies are conducted to define the [A, b, d] model using mixed effect modeling techniques disclosed above.
  • twenty chemicals are used to formulate the mixed effect model relating the molecular descriptors [R, ⁇ , ⁇ , ⁇ , V, and log L] to the IPPSF model parameters
  • model parameters use the apparent molecular descriptors [R', ⁇ ', ⁇ ', ⁇ ', U] and V as used for the ethanol/water exposures.
  • Biological effects of the complex chemical mixture being studied are accounted for in a similar manner using biological co-variates [FTIR shifts, IL-8 release] as disclosed herein.
  • Residual variability is modeled to quantitate unexplained interactions. For example, ionic interactions or chemical reactions between mixture constituents can occur which are detectable in the MCF, but not easily quantifiable or assignable by solvation energy relationships (e.g.
  • Example 4-3 Biomarkers The approach to assessing significant chemical interactions in the
  • IL-8 Release Methods in Keratinocyte Cell Cultures is assessed according to the standardized technique utilized by Allen etal..2000,
  • pig skin is obtained from pigs used to generate IPPSF that have already been euthanatized.
  • the skin is dermatomed at 400 ⁇ m then cut into 2cm 2 pieces, which is floated, dermis side down, on 0.25% trypsin overnight at 4°C.
  • the epidermis is peeled away from the dermis and placed into Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • a single cell suspension is achieved by agitating the epidermal pieces with a pipette and filtering the resultant slurry through a 70 ⁇ m mesh screen.
  • Porcine keratinocytes are plated onto collagen Type I (Collaborative Research Products)-coated 96 well plates overnight in DMEM.
  • the media is replaced by a calcium-free keratinocyte basal media (KBM, Clonetics of San Diego, California, United States of America) supplemented with human epidermal growth factor (0.1 ng/ml), insulin (5 ⁇ g/ml), bovine pituitary extract (0.4%), and 50 ⁇ g/ml gentamicin/50 ng/ml amphotericin-B to create keratinocyte growth media (KGM-2, Clonetics).
  • KBM calcium-free keratinocyte basal media
  • human epidermal growth factor 0.1 ng/ml
  • insulin 5 ⁇ g/ml
  • bovine pituitary extract 0.4%
  • 50 ⁇ g/ml gentamicin/50 ng/ml amphotericin-B 50 ⁇ g/ml gentamicin/50 ng/ml amphotericin-B
  • KGM-2 gentamicin/50 ng/ml amphotericin-B
  • NR neutral red
  • Cytotoxicity is assessed by determining the percentage of viable PEK cells surviving the test chemical/chemical mixture exposure. Cell viability is determined by the NR uptake method. Briefly, 50 ⁇ g/ml NR in KGM-2 (NR medium) are pre-incubated at 37°C overnight and 200 ⁇ l is added to the treated wells after sample collection.
  • a sandwich ELISA assay is used for IL-8.
  • Commercial kits for the detection of porcine IL-8 (Biosource, Camarillo, California, United States of America) are performed as indicated in the manufacturer instructions. The plates were read at 450 nm on a MultiskanTM RC plate reader (Labsystems, Helsinki, Finland). A recombinant porcine IL-8 is diluted to create a standard reference curve.
  • FTIR Spectroscopy is well proven for characterizing aspects of weak interactions, especially, the intermolecular interactions studied herein.
  • Two representative approaches can be employed in assessing a compound's interaction with stratum corneum lipids: (1 ) assessing changes in lipid spectra; or (2) assessing changes in a compounds signature when strongly interacting in a lipid environment. Therefore, FTIR is good second experimental method to verify the results of the MCF technique, and compensate the need for molecular scale understanding the mechanisms of the intermolecular interactions.
  • Vibrational signals of the functional groups of the study compounds are studied in reference solution or film, stratum corneum and epidermis in order to reveal the intermolecular interactions in the percutaneous absorption.
  • the spectrum shift, ⁇ (FTIR shift) is determined for an assessment of lipid induced changes. Four replicates per condition are obtained to get a reliable estimate of shift. If the vibrational signal of a compound is interfered by the skin matrix, deuterated compound can be used.
  • the FTIR equipment is a PE Spectrum 1000 with AutolmageTM microscope (available from PerkinElmer Corporation, Wellesley, Massachusetts, United States of America).
  • An HP 7675 automatic sampler is used to inject 4 ⁇ l of the calibration standard solution, while the membrane-coated fibers are injected manually.
  • the injector is maintained at 280°C for sample vaporization and thermal desorption.
  • Three capillary columns are selected for separation all the calibration compounds and test chemicals (HP-5MS for non-polar and low polar, HP-50 for medium polar and HB-INNOWax for high polar compounds).
  • the separation condition is optimized for resolution and sensitivity.
  • An electronic pressure control is used to maintain a carrier gas flow of 1.00 ml/min helium.
  • the chemicals that permeate into the membrane are qualitatively analyzed in scan-mode.
  • each compound in the complex mixture was accomplished by using HP CHEMSTATIONTM software and matching its fingerprint spectra with a HP MS database.
  • the selected ion monitoring (SIM) mode, and characteristic ions (m/z) are selected for each compound referencing to its spectra acquired experimentally with standard or from MS database. This approach was used to define the MCF technique (Examples 1 and 2 herein above) as well as to assess chemical absorption in porcine skin diffusion cells using the same perfusate composition as the IPPSF (Muhammad et al.. 2003).
  • Frazier JM Goldberg AM. Alternatives to and reduction of animal use in biomedical research, education and testing. Altern. Lab. Anim. 18: 65-74, 1990. Geinoz S, Rey S, Boss G, Bunge A, Guy RH, Carrupt PA, Resit M, Testa B.
  • Monteiro-Riviere NA Inman AO. Characterization of sulfur mustard-induced toxicity by enzyme histochemistry in porcine skin. Toxicology Methods 10:1-16, 2000. Monteiro-Riviere NA. Anatomical Factors Affecting Barrier Function. In
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  • Roberts MS Anissimov YG, Gonsalvez RA. Mathematical Models in Percutaneous Absorption. In Percutaneous Absorption, 3 rd Ed. (Bronaugh RL, Maibach HI eds). Marcel Dekker: New York, pgs. 3-55, 1999. Robinson PJ. Prediction: Simple risk models and overview of dermal risk assessment. In Dermal Absorption and Toxicity Assessment (Roberts MS, Walters KA eds). Marcel Dekker: New York, pgs. 203-229, 1998. S. Agatonovic-Kustrin, R. Beresford, A.P.M. Yusof, J. Pharm. Biomed. Anal. 26(2001)241 -254.
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  • Riviere JE A physiologically relevant pharmacokinetic model of xenobiotic percutaneous absorption utilizing the isolated perfused porcine skin flap (IPPSF). J. Pharm. Sci. 79: 305-31 1 , 1990.
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Abstract

La présente invention concerne un procédé permettant de déterminer un descripteur moléculaire d'absorption pour un candidat composé. Ce procédé comporte plusieurs opérations: obtention d'une solution test comprenant un ou plusieurs candidats composés; mise en contact de la solution test avec une membrane biologique simulée pour fractionner les candidats composés dans la membrane, détectant la présence ou la quantité de ces candidats composés dans la membrane pour une ou plusieurs durées de perméation; et détermination d'un descripteur modulaire d'absorption par la présence ou la quantité de candidats composés dans la membrane pour une ou plusieurs durées de perméation. Dans un aspect, l'invention concerne une membrane imitant la peau, et un ou plusieurs paramètres d'absorption percutanée sont déterminés. Dans un autre aspect, la membrane est disposée sur une fibre. Dans un autre aspect, la solution test est mise en contact avec plusieurs membranes biologiques pour séparer les candidats composés dans les membranes.
PCT/US2003/006756 2002-03-05 2003-03-05 Procede et appareil pour determiner un descripteur moleculaire d'absorption pour un candidat compose WO2003076927A1 (fr)

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